RNase H - LeGriceA full provisional patent application was filed August 30, 2005, on a class of vinylogous ureas which were discovered in the ChemBridge library. Extract hit evaluation is complete, and fractionation of a select set of natural product extracts is underway. All routine secondary evaluation of pure compounds from the various synthetic libraries is complete. We published a paper describing a second lead series of tropolone natural products, and a crystallographic collaboration with Ed Arnold (Rutgers) has succeeded in co-crystallizing one of the vinylogousurea hits with the HIV-1 target. Biacore and ultracentrifugation studies have begun with Inna Goroschkova and Peter Schuck of DBEPS, ORS, NIH. Bob Crouch (NICHD) is pursuing compounds with selectivity for the human RNase H to better understand the functions of this enzyme. An automated calorimeter has been acquired with IATAP funding and used to conduct studies of the binding of hit compounds with the protein target. Initial isothermal titration and differential scanning calorimetry studies have begun on the interaction between RNase H and several candidate compounds identified in the screen. V-ATPase - Khanna/HelmanIn vivo work with a murine osteosarcoma model has been done with Dr. Khanna. To support this, we worked with DTP staff (Stinson, Hollingshead, Alley) to obtain pharmacokinetic data on oximidine III with a cellular bioassay. Preliminary results have shown that therapeutic blood levels can be achieved in mice; however, the invivo osteosarcoma study was negative. Exploration of activity in melanoma models is under consideration.Dr. Porco (BU) is making more oximidine III for further studies, while Yamanouchi Pharmaceutical has recently expressed interest in supplying oximidine III and lobatamide A from fermentation sources. There has been progress with Australian sources from invertebrates, with signing of an MTA with the Australian authorities. It is our goal to better understand V-ATPase as a potential cancer target. We recently correlated cellular sensitivity in the 60-cell panels with RNA expression of certain isoforms of V-ATPase subunits, especially those in the V1 domain. Among other things, this may implicate the V1 domain as the site of action of these compounds. We plan to seek similar data for the osteosarcoma cell lines and to extend this with RT-PCR and RNAi studies. As compound becomes available, we are planning to work with the Guise group (U. VA) on bone biology in osteoclasts with our compounds. We also plan to compare the activity of the compounds in traditional metastasis models such as wound healing and migration for highly metastatic and non-metastatic counterparts in breast and prostate cell lines.HDAC inhibitor screen - HagerScreening of the structural diversity set has been completed.Induction of GFP expression is the endpoint for the HDAC inhibitor screen. The assay was originally intended as a plate reader assay, however, the cells are not bright enough for detection. Consequently, the assay is being conducted using the Discovery-1 imaging system to acquire the images in a 96-well plate format. Hits are identified by qualitative examination of the images. The parental cell line is being used to identify false positive hits resulting from autofluorescent compounds. Low-throughput screening using the structural diversity set has been completed. Fourteen of the 2,080 compounds in the structural diversity set demonstrated some activity in the screen (hit rate 0.67%). Some compounds gave strong GFP induction, while others demonstrated weaker activity. We are in the process of cherry picking the hits and re-confirming the activity found in the primary screen. Screening of additional small chemical libraries has also been completed, with a series of synthetic compounds obtained from Johnny Easmon (U. of Innsbruck) showing consistent activity in the HDAC screen.